The present invention, in some embodiments thereof, relates to the use of amelogenin for enhancing the regeneration of hyaline cartilage, and more specifically articular cartilage.
Cartilage is a mesenchymal tissue; mesenchymal stem cells (MSCs) differentiate to form matrix secreting chondroblasts. Chondroblasts become mature chondrocytes when the matrix encircles them. Depending on the composition of the matrix, cartilage in human body is classified into elastic, fibro-cartilage, fibro-elastic and hyaline cartilage. The gliding surfaces of the synovial joint are covered with a specialized type of hyaline cartilage, termed articular cartilage, which by itself is composed of four zones; superficial, transitional, middle and calcified cartilage zone. Each zone has its unique and characteristic structure of chondrocytes and matrix arrangement, which is crucial for the function of the whole tissue. The superficial zone, the thinnest of all layers, is composed of flattened ellipsoid cells. They lie parallel to the joint surface, and are covered by a thin film of synovial fluid, called ‘lubricin’. This protein is responsible for providing an ultimate gliding surface to the articular cartilage. Chondrocytes in this zone synthesize high concentrations of collagen and low concentrations of proteoglycans, hence, it is the highest water content zone within the articular cartilage. Parallel arrangement of the fibrils are responsible for providing the greatest tensile and shear strength. Disruption of this zone alters the mechanical properties of the articular cartilage and thus contributes to the development of osteoarthritis. This layer also acts as a filter for large macro-molecules, thereby protecting the cartilage from synovial tissue immune system. In the transitional zone the cell density is lower, with predominantly spheroid shaped cells, embedded in abundant extracellular matrix. The large diameter collagen fibers are randomly arranged in this zone. In the middle zone cells are arranged perpendicular to the surface and are spherical in shape. This zone contains the largest diameter of collagen fibrils and the highest concentration of proteoglycans. However, the cell density is lowest in this zone. The calcified cartilage zone is mineralized, contains small volume of cells embedded in a calcified matrix and thus showing a very low metabolic activity. The chondrocytes in this zone express hypertrophic phenotype. These cells are unique since they synthesize type X collagen, responsible for providing important structural integrity and a shock absorber along with the subchondral bone.
Hyaline cartilage provides a low-friction gliding surface, with increased compressive strength and is known to be wear-resistant under normal conditions. Hyaline articular cartilage is aneural, avascular and alymphatic structure. Chondrocytes, the mature cartilage cell (1-5% of its volume), receive their nutrition by diffusion through the matrix. Regeneration of hyaline cartilage is a major scientific challenge; a typical response of tissue to injury follows a cascade of necrosis, inflammation, repair and scar remodeling. The most important determinant of the healing process is the vascular phase of this cascade. Hyaline cartilage, being an avascular structure, lacks the ability to generate this vital response, leading to very low intrinsic reparative capability. When cartilage do heal, fibrocartilage is formed, which lacks the unique structural arrangement and biochemical composition of the hyaline cartilage, hence is inferior clinically and eventually leads to joint degeneration.
Physicians and scientists have sought different ways to repair or regenerate articular surface of synovial joint following traumatic damage or degeneration of the cartilage. The variety of surgical treatments based on different methods aim to increase the quantity of cells capable to differentiate into chondrocytes at the injured site. Penetration of subchondral bone is among the oldest and still the most commonly used method to stimulate regeneration of neo-cartilage. Penetration of subchondral bone plate disrupts the subchondral blood vessels. This leads to the formation of a ‘super clot’ or fibrin clot on the surface of a chondral defect. If the defect is protected from loading at this stage, then primitive bone marrow mesenchymal stem cells migrate into the super clot, to proliferate and differentiate into cells resembling chondrocytes morphologically. A newer modality for repair of osteochondral defects is the use of autologus culture expanded chondrocytes or bone marrow mesenchymal stem cells, implanted into the defect in order to regenerate the tissue. In spite of efforts to produce different treatment methods, none of the above has been able to regenerate a neo-cartilage which is similar in structure and functions to that of a native articular cartilage.
Osteoarthritis (OA) is a chronic degenerative joint disease that progressively causes loss of joint function. The morphologic and biochemical manifestations of OA are; articular cartilage breakdown, subchondral sclerosis, osteophyte formation, bone marrow lesions and alterations of the synovium. The altered biomechanics seen in OA induce and potentiate biochemical changes. Key events occurring in cartilage during the pathogenesis of OA include an imbalance of metabolic and degradative signals. Chondrocytes, as well as synovial cells, of OA patients produce increased levels of inflammatory cytokines, such as interleukin-1β (IL-1b) and tumor necrosis factor-α (TNF-α), which in turn decrease anabolic collagen synthesis and increase catabolic (including matrix metalloproteinases—MMPs) and other inflammatory mediators such as IL-8, IL-6, and prostaglandin E2. In addition, mechanical stress increases nitric oxide production by chondrocytes as well as nitric oxide synthetase expression. These reactive oxygen species have been implicated indirectly in promoting chondrocyte apoptosis, catabolic processes and matrix degradation.
The amelogenins are a major component of the developing extracellular enamel matrix proteins, produced by the ameloblast cells and play a major role in the biomineralization and structural organization of enamel (Robinson et al. 1998). The human amelogenin gene contains 7 exons, which undergo alternative mRNA splicing. The most abundant amelogenin lacks the internal region encoded by exon 4, is termed HX175 in human, which corresponds to isoform M180 in mice. The relatively large number of amelogenin alternatively spliced mRNA translated polypeptides and the fact that amelogenin is expressed in different tissues (calcifying and soft tissues) and of different embryonic origin, possibly reflect different functions of amelogenin.
Amelogenin was shown to be expressed in periodontal ligament (PDL) cells, in long bone cells; osteocytes, osteoblasts and osteoclasts, in cartilage chondrocytes and differentially in growth plate cells. In addition, amelogenin was identified in long bone marrow stromal cells, some of which are multi-potent stem cells (Haze et al. 2007). Furthermore, in the normal uninjured animal, amelogenin expression is increased at sites of high activity and remodeling of ligaments and bones (Haze et al. 2009). Amelogenin expression was also identified in cells of non-mineralizing tissues such as brain and eye in embryonic and postnatal tissues (Deutsch et al. 2006, Gruenbaum-Cohen et al. 2008).
Recombinant human amelogenin has been shown to be beneficial for the treatment of periodontitis (Haze et al. 2009).
International Patent Application WO2011/030185 teaches a cell guiding scaffold which according to one embodiment may comprise amelogenin as one of its active agents for inducing periodontal tissue regeneration. The scaffold may be used for joint ligament regeneration as well.
International Patent Application WO 00/06734 teaches that amelogenin is useful for generation of bone and cartilage. More specifically, this application teaches that it causes an up-regulation in type II collagen and has no effect or negative effect on type I collagen.
U.S. Patent Application Publication Nos. 20100093632 and 20030077291 teach the use of amelogenin for the treatment of inflammatory disorders.
U.S. Patent Application Publication No. 20140073765 teaches the use of amelogenin for the treatment of cartilage injuries.
Additional background material includes European Patent Publication Nos. 0337967, 1862143 and 0053197 and US Patent Application No. 20110003745.